Ketogulonigenium endogenous plasmids

ABSTRACT

The present invention relates, in general, to a genus of bacteria known as  Ketogulonigenium . The present invention further relates to transformed  Ketogulonigenium , and methods of transforming  Ketogulonigenium . The present invention also relates to nucleic acid molecules, and vectors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional application is a divisional application of U.S.application Ser. No. 09/826,191, filed Apr. 5, 2001, now abandoned,which claims the benefit of U.S. provisional Application No. 60/194,627,filed Apr. 5, 2000, the contents of each of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates, in general, to a novel genus of bacteriaknown as Ketogulonigenium. The present invention further relates totransformed Ketogulonigenium, and methods of transformingKetogulonigenium. The present invention also relates to nucleic acidmolecules, and vectors.

2. Background Art

The exploitation of microorganisms to synthesize vitamin C or itschemical pathway intermediates has both economic and ecologicaladvantages.

One key intermediate in vitamin C synthesis is 2-keto-L-gulonic acid(2-KLG), which is easily converted chemically to L-ascorbic acid(vitamin C) by esterification followed by lactonization (Delic, V. etal., “Microbial reactions for the synthesis of vitamin C (L-ascorbicacid,” in Biotechnology of Vitamins, Pigments and Growth Factors,Vandamme, E. J., ed., Elsevier Applied Science (London & New York) pp.299–336 (1989)). Members of a number of bacterial genera have beenidentified that produce 2-KLG from the oxidation of L-sorbose orsorbitol. Such 2-KLG producing genera include the acidogenic,alpha-proteobacteria Gluconobacter and Acetobacter, thegamma-proteobacteria Pseudomonas, Escherichia, Klebsiella, Serratia andXanthmonas and the Gram positive Bacillus, Micrococcus andPseudogluconobacter (Imai, K. et al., U.S. Pat. No. 4,933,289 (1990),Sugisawa, H. et al., “Microbial production of 2-keto-L-gulonic acid fromL-sorbose and D-sorbitol by Gluconobacter melanogenus,” Agric. Biol.Chem. 54:1201–1209 (1990), Yin, G. et al., U.S. Pat. No. 4,935,359(1990), Shirafuji, et al., U.S. Pat. No. 4,876,195 (1989) and Nogami, I.et al., U.S. Pat. No. 5,474,924 (1995)).

To aid in increasing the yield of bacterial products, attempts have beenmade to exploit endogenous plasmids within microorganism strains.(Beppu, T. et al., U.S. Pat. No. 5,580,782 (1996), Fujiwara, A. et al.,U.S. Pat. No. 5,399,496 (1995); Tonouchi et al, U.S. Pat. No. 6,127,174(2000), Hoshino, T. et al., U.S. Pat. No. 6,127,156 (2000)).

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention provides an isolated or purifiednucleic acid molecule comprising a polynucleotide having a nucleotidesequence at least 95% identical to a nucleotide sequence of aKetogulonigenium plasmid replicon found on the endogenous plasmidcontained in NRRL Deposit No. B-21627 and at least one exogenousnucleotide sequence.

Further embodiments of the invention include an isolated or purifiednucleic acid molecule comprising a polynucleotide having a nucleotidesequence at least 90% identical, and more preferably at least 95%, 97%,98% or 99% identical, to any of the above nucleotide sequences, or apolynucleotide which hybridizes under stringent hybridization conditionsto a polynucleotide having a nucleotide sequence as in the above. Thepolynucleotide which hybridizes does not hybridize under stringenthybridization conditions to a polynucleotide having a nucleotidesequence consisting of only A residues or of only T residues.

The present invention relates, in general, to a novel genus of bacteriaknown as Ketogulonigenium. The present invention further relates toKetogulonigenium comprising a transgene (recombinant DNA), comprising anendogenous plasmid. The invention also relates to a method fortransforming Ketogulonigenium comprising conjugative transfer of avector from E. coli to Ketogulonigenium, and to a method fortransforming Ketogulonigenium comprising electroporation.

The invention provides a nucleic acid molecule comprising a nucleotidesequence at least 95% identical to a Ketogulonigenium endogenous plasmidcontained in NRRL Deposit No. B-21627. The invention also provides anucleic acid molecule comprising a polynucleotide having a sequence atleast 95% identical to a Ketogulonigenium replicon found on anendogenous plasmid contained in NRRL Deposit No. B-21627. The inventionfurther provides a vector comprising a nucleic acid molecule comprisinga nucleotide sequence of a Ketogulonigenium replicon found on anendogenous plasmid contained in NRRL Deposit No. B-21627.

Further advantages of the present invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A–1E show the nucleotide (SEQ ID NO:1) sequence of an endogenousplasmid, determined by sequencing of the endogenous plasmid (pADMX6L1),contained in NRRL Deposit No. B-21627. The nucleotide has a sequence ofabout 7029 nucleic acid residues.

FIGS. 2A–2C show the nucleotide (SEQ ID NO:2) sequence of an endogenousplasmid, determined by sequencing of the endogenous plasmid (pADMX6L2),contained in NRRL Deposit No. B-21627. The nucleotide has a sequence ofabout 4005 nucleic acid residues.

FIGS. 3A–3N show the nucleotide (SEQ ID NO:3) sequence of an endogenousplasmid, determined by sequencing of the endogenous plasmid (pADMX6L3),contained in NRRL Deposit No. B-21627. The nucleotide has a sequence ofabout 19,695 nucleic acid residues.

FIGS. 4A–4C show the nucleotide (SEQ ID NO:4) sequence of an endogenousplasmid, determined by sequencing of the endogenous plasmid (pADMX6L4),contained in NRRL Deposit No. B-21627. The nucleotide has a sequence ofabout 4211 nucleic acid residues.

FIGS. 5A–5B show the nucleotide (SEQ ID NO:5) sequence of the repliconon an endogenous plasmid (pADMX6L1), determined by homology of aminoacid sequences encoded by the endogenous plasmid to known replicationproteins, contained in NRRL Deposit No. B-21627. The nucleotide has asequence of about 1456 nucleic acid residues.

FIGS. 6A–6B show the nucleotide (SEQ ID NO:6) sequence of the thereplicon on an endogenous plasmid (pADMX6L2),determined by homology ofamino acid sequences encoded by the endogenous plasmid to knownreplication proteins, contained in NRRL Deposit No. B-21627. Thenucleotide has a sequence of about 2401 nucleic acid residues.

FIGS. 7A–7B show the nucleotide (SEQ ID NO:7) sequence of the thereplicon on an endogenous plasmid (pADMX6L3), determined by homology ofamino acid sequences encoded by the endogenous plasmid to knownreplication proteins, contained in NRRL Deposit No. B-21627. Thenucleotide has a sequence of about 2029 nucleic acid residues.

FIG. 8 shows the amino acid sequence (SEQ ID NO:8) of a replicationprotein encoded by an endogenous plasmid (pADMX6L1) determined from thenucleotide sequence of pADMX6L1 contained in NRRL B-21627. Thepolypeptide has a sequence of about 151 amino acids in length.

FIG. 9 shows the amino acid sequence (SEQ ID NO:9) of a replicationprotein encoded by an endogenous plasmid (pADMX6L2) determined from thenucleotide sequence of pADMX6L2 contained in NRRL B-21627. Thepolypeptide has a sequence of about 466 amino acids in length.

FIG. 10 shows the amino acid sequence (SEQ ID NO:10) of a replicationprotein encoded by an endogenous plasmid (pADMX6L3) determined from thenucleotide sequence of pADMX6L3 contained in NRRL B-21627. Thepolypeptide has a sequence of about 342 amino acids in length.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the ABI Prism 3700). Therefore, as is known in theart for any DNA sequence determined by this automated approach, anynucleotide sequence determined herein may contain some errors.Nucleotide sequences determined by automation are typically at leastabout 90% identical, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of the sequenced DNAmolecule.

Unless otherwise indicated, each “nucleotide sequence” set forth hereinis presented as a sequence of deoxyribonucleotides (abbreviated A, G, Cand T). However, by “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U) where each thymidine deoxynucleotide (T) in the specifieddeoxynucleotide sequence in is replaced by the ribonucleotide uridine(U). For instance, reference to an RNA molecule having the sequence ofSEQ ID NO:1 set forth using deoxyribonucleotide abbreviations isintended to indicate an RNA molecule having a sequence in which eachdeoxynucleotide A, G or C of SEQ ID NO:1 has been replaced by thecorresponding ribonucleotide A, G or C, and each deoxynucleotide T hasbeen replaced by a ribonucleotide U.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, in theendogenous plasmids contained in NRRL Deposit No. B-21627. By “stringenthybridization conditions” is intended overnight incubation at 42° C. ina solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C. Bya polynucleotide which hybridizes to a “portion” of a polynucleotide isintended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30–70 nt of the reference polynucleotide. These are useful asdiagnostic probes and primers.

Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g., the deposited endogenous plasmid), forinstance, a portion 50–750 nt in length, or even to the entire length ofthe reference polynucleotide, also useful as probes according to thepresent invention, as are polynucleotides corresponding to most, if notall, of the nucleotide sequence of the deposited DNA or the nucleotidesequence as shown in FIG. 1 (SEQ ID NO:1). By a portion of apolynucleotide of “at least 20 nt in length,” for example, is intended20 or more contiguous nucleotides from the nucleotide sequence of thereference polynucleotide, (e.g., the deposited DNA or the nucleotidesequence as shown in FIG. 1 (SEQ ID NO:1)). As indicated, such portionsare useful diagnostically either as a probe according to conventionalDNA hybridization techniques or as primers for amplification of a targetsequence by the polymerase chain reaction (PCR), as described, forinstance, in Molecular Cloning, A Laboratory Manual, 2nd. edition,edited by Sambrook, J., Fritsch, E. F. and Maniatis, T., (1989), ColdSpring Harbor Laboratory Press, the entire disclosure of which is herebyincorporated herein by reference.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence may be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence may be inserted intothe reference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in FIG. 1 or to the nucleotide sequence of thedeposited endogenous plasmid can be determined conventionally usingknown computer programs such as the FastA program. FastA does a Pearsonand Lipman search for similarity between a query sequence and a group ofsequences of the same type nucleic acid. Professor William Pearson ofthe University of Virginia Department of Biochemistry wrote the FASTAprogram family (FastA, TFastA, FastX, TFastX and SSearch). Incollaboration with Dr. Pearson, the programs were modified anddocumented for distribution with GCG Version 6.1 by Mary Schultz and IrvEdelman, and for Versions 8 through 10 by Sue Olson.

The present invention provides Ketogulonigenium, comprising a transgene(recombinant DNA) comprising an endogenous Ketogulonigenium plasmid.Preferably, the endogenous Ketogulonigenium plasmid is contained inDeposit No. NRRL B-21627. As used herein, a transgene is defined as atransplanted nucleotide sequence which is exogenous, or non-native, tothe host. An exogenous nucleotide sequence, as used in the currentcontext, is a nucleotide sequence which is not found in Deposit No. NRRLB-21627. Thus, the term exogenous nucleotide sequence is meant toencompass a nucleotide sequence that is foreign to Deposit No. NRRLB-21627, as well as a nucleotide sequence endogenous, or native, toDeposit No. NRRL B-21627 that has been modified. Modification of theendogenous nucleotide sequence may include, for instance, mutation ofthe native nucleotide sequence or any of its regulatory elements. Asused herein, mutation is defined as any change in the wild-type sequenceof genomic or plasmid DNA. An additional form of modification may alsoinclude fusion of the endogenous nucleotide sequence to a nucleotidesequence that is normally not present, in relation to the endogenousnucleotide sequence. The transgene may be regulated by its normalpromoter, or more commonly, by a promoter that normally regulates adifferent gene. The invention also provides a method for producingtransformed Ketogulonigenium, comprising transforming Ketogulonigeniumwith a transgene, comprising, part or all of an endogenousKetogulonigenium plasmid. Preferably, the endogenous Ketogulonigeniumplasmid is contained in Deposit No. NRRL B-21627.

The term replicon as used herein is meant to encompass a DNA sequencecomprising those genes and gene expression control elements such aspromoters and terminators, other DNA sequence features such as shortsequence repeats (iterons), origins of plasmid replication (ori or oriVsites), or other DNA sequence features that are required to support theautonomous replication of a circular DNA molecule in a bacterial host(Chapter 122, pp. 2295–2324, in Escherichia coli and Salmonella:Cellular and Molecular Biology, 2^(nd) Edition, Frederick C. Neidhardt,Ed., ASM Press (1996)). The requirements of a replicon can vary from aslittle as a short ori sequence in the case of plasmids that do notrequire their own replication proteins, to larger sequences containingone or more plasmid-borne replication genes.

The definition of a transformed cell, as used herein, is a cell whereDNA has been inserted into a bacterial cell. The transformation ofKetogulonigenium may be transient or stable. Preferably, the inventionprovides a method for producing stably transformed Ketogulonigenium. Asused herein, a stably transformed cell is a cell wherein a transgene istransmitted to every successive generation. A preferred embodiment ofthe present invention is that Ketogulonigenium is transformed viaelectroporation. An additional preferred embodiment of the presentinvention is that Ketogulonigenium is transformed by the process ofconjugation, including, for instance, bi-parental and tri-parentalconjugation. Conjugation, as used herein, is the process by whichbacteria transfer DNA from a donor cell to a recipient cell throughcell-to cell contact.

The present invention relates to a novel genus of bacteria, comprisingthe ADMX6L strain, designated as Ketogulonigenium. The present inventorshave discovered novel strains of bacteria, not belonging to any knowngenera, that produce 2-keto-L-gulonic acid from sorbitol.

Ketogulonigenium (Ke.to.gu.lo.ni.gen'.i.um. M. L. n. acidumketogulonicum ketogulonic acid; Gr. V. gennaio to produce; M. L. n.ketogulonigenium ketogulonic acid producing) is gram negative,facultatively anaerobic, motile or non-motile, has ovoid to rod-shapedcells, 0.8–1.3 μm long, 0.5–0.7 μm in diameter, with tapered ends,occurring as single cells, pairs and occasionally short chains. Somestrains form elongated cells (up to 30 μm in length) on TSB. Flagellaand fimbrae have been observed. Colonies are tan colored, smooth,circular, entire, raised to convex, 1–2 mm in diameter with a diffusablebrown pigment after 48 hrs incubation. Oxidase and catalase reactionsare positive. Optimum temperature range is 27 to 31° C., optimum pHrange is 7.2 to 8.5 and optimum Na⁺ concentration is 117–459 mM.Chemoorganotrophic. Carbon sources utilized include arabinose,cellobiose, fructose, glucose, glycerol, inositol, lactose, maltose,mannitol, mannose, rhamnose, sorbitol, sorbose, sucrose, trehalose,pyruvate and succinate. Favored carbon sources are inositol, mannitol,glycerol, sorbitol, lactose and arabinose. All strains examined produce2-keto-L-gulonic acid from L-sorbose. Major cellular fatty acids are16:0 and 18:1 ω7c/ω9t/ω12t and the mol % DNA G+C is 52.1 to 54.0percent. Small subunit rDNA sequence analysis place this genus in thealpha subgroup of the Proteobacteria. All strains isolated in thepresent study group originated in soil.

DNA reassociation studies divide the genus into two species. K. vulgaraeand K. robustum, of which K. vulgarae is the designated type species. Agroup of bacteria having the above-mentioned properties does not belongto any known genera as described in Bergey's Manual of SystematicBacteriology, and therefore belongs to a new genus.

Strain ADMX6L of Ketogulonigenium was isolated and deposited at theAgricultural Research Service Culture Collection (NRRL), 1815 NorthUniversity Street, Peoria, Ill. 61604, USA, on Oct. 1, 1996, under theprovisions of the Budapest Treaty, and assigned accession numbers NRRLB-21627.

The present invention provides a method for conjugative transfer of avector from E. coli to Ketogulonigenium comprising culturing the E. coliwith the Ketogulonigenium under such conditions such that the E. colitransfers the vector to the Ketogulonigenium. The method of conjugativetransfer relies on the ability of the vector to replicate in bothorganisms, and thus requires that the vector contain replicons that arefunctional in both organisms. A replicon is a nucleotide sequence,typically several hundred to several thousand base pairs long, that isvital to plasmid DNA replication. Preferably, the method comprises usingany vector that contains a replicon that is functional in E. coli, aswell a replicon that is functional in Ketogulonigenium. More preferably,the method of the invention comprises the vectors pDELIA8 and pXH2/K5.Given that the preferred method comprises using any vector that containsa replicon that is functional in E. coli, as well as a replicon that isfunctional in Ketogulonigenium, it would also be possible to transfer avector, via conjugation, from Ketogulonigenium to E. coli.

The present invention also provides a method for transformingKetogulonigenium comprising inserting a vector into the Ketogulonigeniumthrough the process of electroporation. Preferably, the vector used inelectroporation of Ketogulonigenium is pMF1014-α.

The present invention provides isolated or purified nucleic acidmolecules comprising the polynucleotides, or their complements, ofendogenous plasmids that have been isolated and purified from a strain,NRRL Deposit No. B-21627 (ADMX6L), of this novel genus. Four endogenousplasmids have been isolated from strain NRRL Deposit No. B-21627. Thenucleotide sequence shown in FIG. 1 (SEQ ID NO:1) is the nucleotidesequence of plasmid pADMX6L1 as determined by automated sequencing. Thenucleotide sequence shown in FIG. 2 (SEQ ID NO:2) is the nucleotidesequence of plasmid pADMX6L2 as determined by automated sequencing. Thenucleotide sequence shown in FIG. 3 (SEQ ID NO:3) is the nucleotidesequence of plasmid pADMX6L3 as determined by automated sequencing. Thenucleotide sequence shown in FIG. 4 (SEQ ID NO:4) is the nucleotidesequence of plasmid pADMX6L4 as determined by automated sequencing.

The endogenous plasmids contained within NRRL B-21627 (ADMX6L) have beenisolated and ligated into pUC19 or pJND1000. Specifically, plasmidpADMX6L1, corresponding to SEQ ID NO:1, and pJND1000 are digested withBamHI and ligated to each other using T4 ligase. The pXB1 plasmidconstruct was then introduced into E. coli DH5αMCR and the culturecollection was deposited at the Agricultural Research Service CultureCollection (NRRL), 1815 North University Street, Peoria, Ill. 61604,USA, on Feb. 23, 2001, under the provisions of the Budapest Treaty, andassigned accession numbers NRRL B-30418. Plasmid pADMX6L2, correspondingto SEQ ID NO:2, and pUC19 were digested with HinDIII and ligated to oneanother using T4 ligase. The pXH2 plasmid construct was then introducedinto E. coli DH5αMCR and the culture collection was deposited at theAgricultural Research Service Culture Collection (NRRL), 1815 NorthUniversity Street, Peoria, Ill. 61604, USA, on Feb. 23, 2001, under theprovisions of the Budapest Treaty, and assigned accession numbersNRRLB-30419. Plasmid pADMX6L4, corresponding to SEQ ID NO:4, and pUC19are digested with SspI and SmaI, respectively, and ligated to oneanother using T4 ligase. The pXB4 plasmid construct was then introducedinto E. coli DH5αMCR and the culture collection is deposited at theAgricultural Research Service Culture Collection (NRRL), 1815 NorthUniversity Street, Peoria, Ill. 61604, USA, on Mar. 12, 2001, under theprovisions of the Budapest Treaty, and assigned accession number NRRLB-30435.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the ABI Prism 3700). Therefore, as is known in theart for any DNA sequence determined by this automated approach, anynucleotide sequence determined herein may contain some errors.Nucleotide sequences determined by automation are typically at leastabout 90% identical, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of the sequenced DNAmolecule. The actual sequence can be more precisely determined by otherapproaches including manual DNA sequencing methods well known in theart.

Unless otherwise indicated, each “nucleotide sequence” set forth hereinis presented as a sequence of deoxyribonucleotides (abbreviated A, G, Cand T).

However, by “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U) where each thymidine deoxynucleotide (T) in the specifieddeoxynucleotide sequence is replaced by the ribonucleotide uridine (U).For instance, reference to an RNA molecule having the sequence of SEQ IDNO:1 set forth using deoxyribonucleotide abbreviations is intended toindicate an RNA molecule having a sequence in which each deoxynucleotideA, G or C of SEQ ID NO:1 has been replaced by the correspondingribonucleotide A, G or C, and each deoxynucleotide T has been replacedby a ribonucleotide U.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

One aspect of the invention provides an isolated or purified nucleicacid molecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence in SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, and SEQ ID NO:7; (b) a nucleotide sequence of a plasmid containedin NRRL Deposit No.

B-30418, NRRL Deposit No. B-30419, and NRRL Deposit No. B-30435; and (c)a nucleotide sequence complementary to any of the nucleotide sequencesin (a) or (b) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 97%, 98% or 99%identical, to any of the Ketogulonigenium nucleotide sequences in (a),(b) or (c) above, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide having a nucleotidesequence identical to the Ketogulonigenium portion of a nucleotidesequence in (a), (b) or (c), above. The polynucleotide which hybridizesdoes not hybridize under stringent hybridization conditions to apolynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues.

A polynucleotide having a nucleotide sequence at least, for example, 95%“identical” to a reference nucleotide sequence is intended to mean thatthe nucleotide sequence of the polynucleotide is identical to thereference sequence except that the polynucleotide sequence may includeup to five point mutations per each 100 nucleotides of the referencenucleotide sequence. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 97%, 98% or 99% identical to, for instance, thenucleotide sequences shown in FIG. 1, 2, 3, 4, 5, 6, or 7, or to thenucleotide sequences of the deposited plasmids can be determinedconventionally using known computer programs such as the FastA program.FastA does a Pearson and Lipman search for similarity between a querysequence and a group of sequences of the same type nucleic acid.Professor William Pearson of the University of Virginia Department ofBiochemistry wrote the FASTA program family (FastA, TFastA, FastX,TFastX and SSearch). In collaboration with Dr. Pearson, the programswere modified and documented for distribution with GCG Version 6.1 byMary Schultz and Irv Edelman, and for Versions 8 through 10 by SueOlson.

The present application is directed to nucleic acid molecules at least95%, 97%, 98% or 99% identical to the nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1), FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:3), andFIG. 4 (SEQ ID NO:4); or to the Ketogulonigenium portion of the nucleicacid sequence of the deposited plasmids.

The present application is also directed to nucleic acid molecules atleast 95%, 97%, 98% or 99% identical to the nucleic acid sequences shownin FIG. 5 (SEQ ID NO:5), FIG. 6 (SEQ ID NO:6), and FIG. 7 (SEQ ID NO:7).

One aspect of the invention provides an isolated nucleic acid moleculecomprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide of a nucleicacid molecule of the invention described above, for instance, in theplasmid contained in NRRL B-30418, NRRL B-30419, or NRRL B-30435, or inSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, or SEQ ID NO:7. By “stringent hybridization conditions” isintended overnight incubation at 42° C. in a solution comprising: 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20μg/ml denatured, sheared salmon sperm DNA, followed by washing thefilters in 0.1×SSC at about 65° C. By a polynucleotide which hybridizesto a “portion” of a polynucleotide is intended a polynucleotide (eitherDNA or RNA) hybridizing to at least about 15 nucleotides (nt), and morepreferably at least about 20 nt, still more preferably at least about 30nt, and even more preferably about 30–70 nt of the referencepolynucleotide. These are useful as diagnostic probes and primers.

Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotides (e.g., the deposited endogenous plasmids), forinstance, a portion 15–750 nt in length, or even to the entire length ofthe reference polynucleotide, are also useful as probes according to thepresent invention, as are polynucleotides corresponding to most, if notall, of the Ketogulonigenium portion of the nucleotide sequence of thedeposited DNA or the nucleotide sequence as shown in FIGS. 1 (SEQ IDNO:1), 2 (SEQ ID NO:2), 3 (SEQ ID NO:3), 4 (SEQ ID NO:4), 5 (SEQ IDNO:5), 6 (SEQ ID NO:6), and 7 (SEQ ID NO:7). A portion of apolynucleotide of, at least 15 nt in length, for example, is intended tomean 15 or more contiguous nucleotides from the nucleotide sequence ofthe reference polynucleotide, (e.g., the deposited DNA or the nucleotidesequences as shown in FIGS. 1 (SEQ ID NO:1), 2 (SEQ ID NO:2), 3 (SEQ IDNO:3), 4 (SEQ ID NO:4), 5 (SEQ ID NO:5), 6 (SEQ ID NO:6), 7 (SEQ IDNO:7)). As indicated, such portions are useful diagnostically either asa probe according to conventional DNA hybridization techniques or asprimers for amplification of a target sequence by the polymerase chainreaction (PCR), as described, for instance, in Molecular Cloning, ALaboratory Manual, 2nd. edition, edited by Sambrook, J., Fritsch, E. F.and Maniatis, T., (1989), Cold Spring Harbor Laboratory Press, theentire disclosure of which is hereby incorporated herein by reference.

One embodiment of the present invention is a vector comprising thenucleic acid molecules contained in the group consisting of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, corresponding to endogenousplasmids contained in Deposit No. NRRL B-21627 (ADMX6L). A preferredembodiment of the present invention is that polynucleotides of interestcan be joined to the nucleic acid molecules of the present invention,which may contain a selectable marker. An additional preferredembodiment is that the vectors comprising the nucleic acid moleculesalso contain a transcription terminator, a promoter and a polylinkersite. As used herein, a polylinker site is defined to be a discreteseries of restriction endonuclease sites that occur between the promoterand the terminator. The vector can optionally contain its nativeexpression vector and/or expression vectors which include chromosomal-,and episomal-derived vectors, e.g., vectors derived from bacterialexogenous plasmids, bacteriophage, and vectors derived from combinationsthereof, such as cosmids and phagemids.

A DNA insert of interest should be operatively linked to an appropriatepromoter, such as its native promoter or a host-derived promoter, thephage lambda P_(L) promoter, the phage lambda P_(R) promoter, the E.coli lac promoters, such as the lacI and lacZ promoters, trp and tacpromoters, the T3 and T7 promoters and the gpt promoter to name a few.Other suitable promoters will be known to the skilled artisan.

The expression constructs will preferably contain sites fortranscription initiation, termination and, in the transcribed region, aribosome binding site for translation. The coding portion of the maturetranscripts expressed by the constructs can include a translationinitiating codon at the beginning and a termination codon appropriatelypositioned at the end of the coding sequence to be translated.

As indicated, the expression vectors will preferably include at leastone marker capable of being selected or screened for. Preferably theselectable marker comprises a nucleotide sequence which confersantibiotic resistance in a host cell population. Such markers includeamikacin, ampicillin, chloramphenicol, erythromycin, gentamicin,kanamycin, penicillin, spectinomycin, streptomycin, or tetracyclineresistance genes. Other suitable markers will be readily apparent to theskilled artisan.

The translated polypeptide encoded by the DNA in the vector may beexpressed in a modified form, such as a fusion protein, and may includenot only secretion signals, but also additional heterologous functionalregions. Thus, for instance, a region of additional amino acids,particularly charged amino acids, may be added to the N-terminus of thepolypeptide to improve stability and persistence in the host cell,during purification, or during subsequent handling and storage. Also,peptide moieties may be added to the polypeptide to facilitatepurification.

The translated protein encoded by the DNA contained in the vector can berecovered and purified from recombinant cell cultures by well-knownmethods including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification.

The present invention also provides vectors comprising nucleic acidmolecules comprising a nucleotide sequence of a replicon found on anendogenous plasmid contained in Deposit No. NRRL B-21627. Preferably,the vector of the present invention comprises a replicon selected fromthe group of nucleotide sequences comprising SEQ ID NO:5, SEQ ID NO:6and SEQ ID NO:7. The replicon is a nucleotide sequence that is typicallyseveral hundred to several thousand base pairs long and encodesfunctions controlling the replication of plasmid DNA. The nucleotidesequence in FIG. 5 (SEQ ID NO:5) is the nucleotide sequence comprising areplicon on plasmid pADMX6L1. The nucleotide sequence in FIG. 6 (SEQ IDNO:6) is the nucleotide sequence comprising a replicon on plasmidpADMX6L2. The nucleotide sequence in FIG. 7 (SEQ ID NO:7) is thenucleotide sequence comprising a replicon on plasmid pADMX6L3.

The present invention further provides a vector which has an repliconthat is functional in Escherichia coli (E. coli) as well as a repliconthat is functional in Ketogulonigenium. The present invention alsoprovides a vector which has a replicon that is functional inKetogulonigenium, as well as a replicon that is functional in any of thegenera comprising the group consisting of Acetobacter, Corynebacterium,Rhodobacter, Paracoccus, Roseobacter, Pseudomonas, Pseudogluconobacter,Gluconobacter, Serratia, Mycobacterium, Streptomyces and Bacillus.Utilizing the fact that the vector comprises a functional replicon inKetogulonigenium, and preferably also comprises a replicon functional inE. coli, the present invention provides for a transformed cell of thegenus Ketogulonigenium, comprising the vector. The present inventionalso provides for a transformed E. coli, comprising the vector. E. coliis known to be an efficient host for amplification of a vector DNA andmanipulation of recombinant DNA by simple and rapid methods. On theother hand, Ketogulonigenium can be used as a host for expression ofKetogulonigenium genes. Since the vectors of the present invention aresuch functional constructs, they enable cloning of certain genes ofKetogulonigenium in E. coli and thereafter the effective expression ofthe genes in Ketogulonigenium. Furthermore, it is favorable that suchfunctional constructs also contain a DNA region necessary for conjugaltransfer (mob site). Hence the vectors of the present invention canfirst be assembled in E. coli and then directly introduced intoKetogulonigenium by conjugal mating without isolation of plasmid DNAfrom E. coli.

The present invention further relates to (a) nucleic acid sequencescomprising at least 95% functional homology with those encodingpolypeptides derived from the four endogenous plasmids fromKetogulonigenium strain ADMX6L, and (b) to amino acid sequencescomprising at least 95% functional homology with those encoded withinthe plasmids. The invention also relates to nucleic acid sequencescomprising at least 95% functional homology with, pADMX6L1 rep ORF(bases 2255–2710 of FIG. 1 (SEQ ID NO:1), pADMX6L2 rep ORF (reversecompliment of bases 3960–2562 of FIG. 2 (SEQ ID NO:2), and pADMX6L3 repORF (reverse compliment of bases 5031–4003 of FIG. 3 (SEQ ID NO:3)). Theinvention further relates to amino acid sequences comprising at least95% functional homology with those set forth in FIG. 8, 9 or 10.

Methods used and described herein are well known in the art and are moreparticularly described, for example, in R. F. Schleif and P. C. Wensink,Practical Methods in Molecular Biology, Springer-Verlag (1981); J. H.Miller, Experiments in Molecular Genetics, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1972); J. H. Miller, A Short Course inBacterial Genetics, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1992); M. Singer and P. Berg, Genes & Genomes, UniversityScience Books, Mill Valley, Calif. (1991); J. Sambrook, E. F. Fritschand T. Maniatis, Molecular Cloning: A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); P. B.Kaufman et al., Handbook of Molecular and Cellular Methods in Biologyand Medicine, CRC Press, Boca Raton, Fla. (1995); Methods in PlantMolecular Biology and Biotechnology, B. R. Glick and J. E. Thompson,eds., CRC Press, Boca Raton, Fla. (1993); P. F. Smith-Keary, MolecularGenetics of Escherichia coli, The Guilford Press, New York, N.Y. (1989);Plasmids: A Practical Approach, 2nd Edition, Hardy, K. D., ed., OxfordUniversity Press, New York, N.Y. (1993); Vectors: Essential Data,Gacesa, P., and Ramji, D. P., eds., John Wiley & Sons Pub., New York,N.Y. (1994); Guide to Electroporation and electrofusions, Chang, D., etal., eds., Academic Press, San Diego, Calif. (1992); PromiscuousPlasmids of Gram-Negative Bacteria, Thomas, C. M., ed., Academic Press,London (1989); The Biology of Plasmids, Summers, D. K., BlackwellScience, Cambridge, Mass. (1996); Understanding DNA and Gene Cloning: AGuide for the Curious, Drlica, K., ed., John Wiley and Sons Pub., NewYork, N.Y. (1997); Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, Rodriguez, R. L., et al., eds., Butterworth, Boston, Mass.(1988); Bacterial Conjugation, Clewell, D. B., ed., Plenum Press, NewYork, N.Y. (1993); Del Solar, G., et al., “Replication and control ofcircular bacterial plasmids,” Microbiol. Mol. Biol. Rev. 62:434–464(1998); Meijer, W. J., et al., “Rolling-circle plasmids from Bacillussubtilis: complete nucleotide sequences and analyses of genes ofpTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with relatedplasmids from gram-positive bacteria,” FEMS Microbiol. Rev. 21:337–368(1998); Khan, S. A., “Rolling-circle replication of bacterial plasmids,”Microbiol. Mol. Biol. Rev. 61:442–455 (1997); Baker, R. L., “Proteinexpression using ubiquitin fusion and cleavage,” Curr. Opin. Biotechnol.7:541–546 (1996); Makrides, S. C., “Strategies for achieving high-levelexpression of genes in Escherichia coli,” Microbiol. Rev. 60:512–538(1996); Alonso, J. C., et al., “Site-specific recombination ingram-positive theta-replicating plasmids,” FEMS Microbiol. Lett.142:1–10 (1996); Miroux, B., et al., “Over-production of protein inEscherichia coli: mutant hosts that allow synthesis of some membraneprotein and globular protein at high levels,” J. Mol. Biol. 260:289–298(1996); Kurland, C. G., and Dong, H., “Bacterial growth inhibited byoverproduction of protein,” Mol. Microbiol. 21:1–4 (1996); Saki, H., andKomano, T., “DNA replication of IncQ broad-host-range plasmids ingram-negative bacteria,” Biosci. Biotechnol. Biochem. 60:377–382 (1996);Deb, J. K., and Nath, N., “Plasmids of corynebacteria,” FEMS Microbiol.Lett. 175:11–20 (1999); Smith, G. P., “Filamentous phages as cloningvectors,” Biotechnol. 10:61–83 (1988); Espinosa, M., et al., “Plasmidrolling cicle replication and its control,” FEMS Microbiol. Lett.130:111–120 (1995); Lanka, E., and Wilkins, B. M., “DNA processingreaction in bacterial conjugation,” Ann. Rev. Biochem. 64:141–169(1995); Dreiseikelmann, B., “Translocation of DNA across bacterialmembranes,” Microbiol. Rev. 58:293–316 (1994); Nordstrom, K., andWagner, E. G., “Kinetic aspects of control of plasmid replication byantisense RNA,” Trends Biochem. Sci. 19:294–300 (1994); Frost, L. S., etal., “Analysis of the sequence gene products of the transfer region ofthe F sex factor,” Microbiol. Rev. 58:162–210 (1994); Drury, L.,“Transformation of bacteria by electroporation,” Methods Mol. Biol.58:249–256 (1996); Dower, W. J., “Electroporation of bacteria: a generalapproach to genetic transformation,” Genet. Eng. 12:275–295 (1990); Na,S., et al., “The factors affecting transformation efficiency ofcoryneform bacteria by electroporation,” Chin. J. Biotechnol. 11:193–198(1995); Pansegrau, W., “Covalent association of the traI gene product ofplasmid RP4 with the 5′-terminal nucleotide at the relaxation nicksite,” J. Biol. Chem. 265:10637–10644 (1990); and Bailey, J. E.,“Host-vector interactions in Escherichia coli,” Adv. Biochem. Eng.Biotechnol. 48:29–52 (1993).

EXAMPLES

The following examples are illustrative only and are not intended tolimit the scope of the invention as defined by the appended claims.

Example 1 Preparation of Plasmid DNA from Ketogulonigenium and E. coliStrains.

A portion of a frozen culture of Ketogulonigenium robustum ADMX6L (NRRLB-12627) was seeded into 10 ml X6L medium (2% mannitol, 1% yeastextract, 1% soytone, 0.5% malt extract, 0.5% NaCl, 0.25% K₂HPO₄, pH 7.8)and grown overnight at 30° C. A portion of a frozen culture ofEscherichia coli transformed with pJND1000 was seeded into 10 ml ofLuria Broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) and grownovernight at 37° C. Both cultures were used to prepare plasmid DNA.

DNA was isolated using the Promega Wizard Plus Midipreps DNAPurification System (Madison, Wis.). The culture was centrifuged at10,000×g for 10 minutes at 4° C. in a Sorval RC-5B centrifuge using theSS-34 rotor. The pellet was suspend in 3 ml of 50 mM Tris-HCl, pH 7.5,10 mM EDTA, 100 μg/ml RnaseA. Three ml of cell lysis solution (0.2MNaOH, 1% SDS) was added and mixed by inverting the tube, then three mlof neutralization solution (1.32M potassium acetate, pH4.8) was addedand mixed by inverting the tube. The lysate was centrifuged at 14,000×gfor 15 minutes at 4° C. in a SS-34 rotor, then the supernatant wascarefully decanted to a new centrifuge tube. Ten ml of resuspensionresin (40% isopropanol, 4.2M guanidine hydrochloride) was added to thesupernatant fluid and mixed by swirling, then the mixture wastransferred into the Promega Wizard Midicolumn, which was connected avacuum manifold. Vacuum was applied to pull the resin/DNA mixturecompletely into the midicolumn. The column was washed twice with 15 mlof column wash solution (95% ethanol, 80 mM potassium acetate, 8.3 mMTris-HCl, pH 7.5, 40 μM EDTA. The reservoir was removed from themidicolumn with a scissors, then the column was placed in amicrocentrifuge tube and centrifuged at 10,000×g for 2 minutes to removeany residual solution. The midicolumn was transferred to a newmicrocentrifuge tube and 300 μl of sterile dH₂O was applied. The tubewas microcentrifuged at 10,000×g for 20 seconds to elute to the DNA intosolution. A similar procedure would be employed for other plasmids fromKetogulonigenium or E. coli, except that choice and concentration ofselective antibiotics would be altered as suitable for the plasmid beingisolated.

Example 2 Transformation of a Ketogulonigenium Host With a Plasmid UsingElectroporation

Ketoguonigenium robustum strain ADMX6L (NRRL B-21627) was transformedwith plasmid pMF1014-α using the electroporation method. PlasmidpMF1014-α was described in a Ph.D. thesis at MIT (M. T. Follettie, “DNATechnology for Corynebacterium glutamicum: Isolation andCharacterization of Amino Acid Biosynthetic Genes,” Ph.D. Dissertation,Massachusetts Institute of Technology, Cambridge, Mass. (1989)).pMF1014α encodes kanamycin resistance and can replicate and bemaintained in both E. coli and in Ketogulonigenium.

Competent Ketogulonigenium cells were prepared by seeding a singlecolony of ADMX6L into 10 ml of X6L medium (1% soytone, 1% yeast extract,0.5% malt extract, 0.5% NaCl, 0.25% K₂HPO₄, 2% mannitol, pH 7.8). Theculture was shaken at 300 rpm at 30 C until reaching an optical densityof 0.8 units at 600 nm wavelength. Five ml of this culture was used toseed 500 ml of fresh X6L medium in a 2 L baffled erlenmeyer flask, whichwas shaken at 300 rpm at 30° C. until reaching an optical density of 0.8units. The culture was chilled in an ice-water bath 10 minutes, thentransferred to a pre-chilled centrifuge bottle and centrifuged at 5,000rpm in a Sorvall SC-RB Refrigerated Centrifuge for 15 minutes at 4° C.Cells were maintained at 2–4° C. for all steps to follow. Thesupernatent was decanted and the cell pellet suspended in 5 ml ice-coldMilli-Q water, then additional cold Milli-Q water was was added to avolume of 500 ml. The cells were centrifuged as before, then rewashed in500 ml of Milli-Q water as before and recentrifuged. The twice-washedcell pellet was suspended in 40 ml of ice-cold 10% glycerol thencentrifuged again as before. The supernatent was decanted, then thecells were suspended in a volume of chilled 10% glycerol approximatelyequal to the volume of the cell pellet. The competent Ketogulonigeniumcells were aliquoted to microcentrifuge tubes (40 μl per tube) andstored at −80° C.

A BioRad “Gene Pulser II” electroporator device was set to 1.5 kV, 25uF. The pulse controller was set to 200 Ohms. One μl of pMF1014-α DNAprepared as in Example 1 was added to 40 μl of thawed chilled competentKetogulonigenium cells on ice. The cell-DNA mixture was transferred to apre-chilled electroporation cuvette, which was then transferred to theelectroporation device and the pulse was applied. One ml of X6L mediumwas added to the cuvette, then the mixture was removed, transferred to a10-ml test tube, and incubated for 2 hours with orbital shaking at 300rpm and 30° C. The incubated cells (approximately 1.4 ml) were placed ina microcentrifuge tube and spun at 13,000 rpm for 2 minutes, after which0.9 ml of clear supernatent was carefully removed. The cell pellet wassuspended in the remaining supernatent, then the cells were spread ontothe surface of an X6L medium agar plate (1.2% Difco Bacto Agar)containing 50 μg/ml of kanamycin, and the plate was incubated for 2–3days at 30° C. Colonies that formed on this plate were Ketogulonigeniumtransformed with plasmid vector pMF1014-α.

Example 3 Transfer of Plasmids From E. coli to Ketogulonigenium, DepositNo. NRRL B-21627 by Bi-Parental or Tri-Parental Mating

(A) Bi-parental Mating

Plasmid pDELIA8 was transferred into Ketogulonigenium from E. coli byconjugation. To make pDELIA8, the AflIII-SphI fragment from plasmidpFD288 (GenBank Accession No. U30830), which contains the cloned mobregion of plasmid RK2 (GenBank Accession No. L27758), was isolated fromagarose gels and ligated to the large AflIII-SphI fragment from plasmidpUC19 (GenBank Accession No. M77789) to make the intermediate plasmidpUC19/oriT. pUC19/oriT DNA was digested with SspI and DraI and the large(2527 bp) fragment was purified from an agarose gel. Plasmid pMF1014-α(M. T. Follettie, “DNA Technology for Corynebacterium glutamicum:Isolation and Characterization of Amino Acid Biosynthetic Genes”, Ph.D.Dissertation, Massachusetts Institute of Technology, Cambridge, Mass.(1989)) was digested with BamHI and PstI, made blunt ended with Klenowfragment, and the large fragment was isolated from an agarose gel. Thegel purified, blunt-ended fragments from pUC19/oriT and pMF1014-α wereligated together to make pDELIA8. pDELIA8 carries a gene for kanamycinresistance, a plasmid replication gene, the replicon that is functionalin E. coli and in Ketogulonigenium, lacZα and a polylinker.

A 5 ml culture of E. coli S17-1 (ATCC 47055) was transformed withpDELIA8 using calcium chloride mediated transformation. TheS17-7/pDELIA8 strain was grown in Luria Broth containing 50 μg/mlkanamycin at 37° C. overnight. Ketogulonigenium robustum strain ADMX6L01was grown in X6L medium containing 50 μg/ml of nalidixic acid at 30° C.overnight. ADMX6L01 is a nalidixic acid resistant mutant of strainADMX6L, derived by conventional mutagenesis of ADMX6L followed byselection of strains showing resistance to 50 μg/ml of nalidixic acid.

Fifty microliters of the fresh S17-1/pDELIA8 culture was transferredinto 3 ml of Luria Broth containing 50 μg/ml of kanamycin and 5 μg/ml oftetracycline and grown at 37° C. until reaching an OD₆₀₀ of 0.2 to 0.4absorption units at 600 nm. Two hundred microliters of the freshADMX6L01 culture was transferred into 3 ml of X6L medium containingnalidixic acid and grown at 30° C. until reaching an OD₆₀₀ of 0.8absorption units. 400 μl of S17-1/pDELIA8 culture was placed in a 1.5 mlmicrocentrifuge tube and microcentrifuged for 2 minutes. The pellet wasresuspended in X6L medium without antibiotics and spun down again. Thispellet was resuspended in 1 ml of the fresh ADMX6L01 culture to achievea suspension of mixed cells. The mixed cells were centrifuged to apellet, resuspended in 1 ml of fresh X6L medium without antibiotics,then pelleted and washed once again in 1 ml of X6L medium. The finalpellet of mixed cells was then resuspended in 100 μl of X6L mediumwithout antibiotics and spotted onto a a sterilized GN-6 metrical 0.45μm×25 mm filter (Gelman Sciences, Product No.63068) resting on thesurface of an X6L agar medium plate without antibiotics, which was thenincubated overnight at 30° C. The cell biomass on the filter wasresuspended in 3 ml of X6L medium and 50 μl of this suspension wasplated onto X6L agar medium containing kanamycin and nalidixic acid atconcentration 50 μg/ml each. Colonies that formed on this plate after2–3 days incubation at 30° C. were Ketogulonigenium transconjugantstransformed with pDELIA8.

(B) Tri-parental Mating

A 5 ml culture of E. coli HB101 (ATCC33694) harboring plasmid pDELIA8was grown in Luria Broth (LB) containing 50 μg/ml of kanamycin at 37degrees overnight. A 5 ml culture of E. coli HB101 harboring plasmid RP1(GenBank Accession No. L27758) was grown in Luria Broth containing 5μg/ml of tetracycline at 37 degrees overnight. A 5 ml culture ofKetogulonigenium robustum ADMX6L01 was grown in X6L medium containing 50μg/ml of nalidixic acid at 30 degrees overnight. Fifty microliters eachof the fresh E. coli HB101/pDELIA8 and HB101/RP1 cultures weretransferred to 3 ml of LB with 50 μg/ml of kanamycin or 5 μg/ml oftetracycline, respectively, and were grown to an OD_(600nm) of 0.2 to0.4. Two hundred microliters of the fresh Ketogulonigenium robustumADMX6L01 culture was transferred to 3 ml of X6L medium containing 50μg/ml of nalidixic acid and grown to an OD_(600nm) of 0.8. Four hundredmicroliters each of the E. coli cultures was combined and pelleted in a1.5 ml microfuge tube, then decanted, resuspended in 1 ml of X6L mediumwithout antibiotics and spun down again. The supernatant was removed andthe E. coli pellet was resuspended in 1 ml of the fresh ADMX6L01culture. The three-strain mixture was centrifuged to a pellet, thenresuspended again 1 ml of X6L medium without antibiotics andrecentrifuged. The pellet from this wash was resuspended in 100 μl ofX6L medium without antibiotics and spotted onto a sterilized GN-6metrical 0.45 μm×25 mm filter (Gelman Sciences, Product No. 63068)resting on the surface of an X6L agar medium plate without antibiotics,which was then incubated overnight at 30° C. The cell biomass on thefilter was resuspended in 3 ml of X6L medium and 50 μl of thissuspension was plated onto X6L agar medium containing kanamycin andnalidixic acid at concentration 50 μg/ml each. Colonies that formed onthis plate after 2–3 days incubation at 30° C. were Ketogulonigeniumtransconjugants transformed with plasmid pDELIA8.

Example 4 Construction of Plasmid Vector pJND1000.

pJND1000 was constructed from segments of plasmids pUC19 (GenBankAccession No. M77789), pUC4K (GenBank Accession No. X06404), pFD288(GenBank Accession No. U30830), and pFC5 (David M. Lonsdale et. al.,“pFC1 to pFC7: A novel family of combinatorial cloning vectors.”, PlantMolecular Biology Reporter 13(4):343–345 (1995)).

The ampicillin resistance gene (amp^(R)) was removed from pUC19 bydigesting pUC19 DNA with restriction enzymes DraI and SspI, separatingthe 1748 bp vector fragment from the smaller amp^(R) fragment and otherfragments by gel electrophoresis, then recovering the 1748 bp fragmentfrom a gel slice. A kanamycin resistance gene (kan^(R)) fragment frompUC4K was prepared by digesting pUC4K with restriction enzyme PstI,treating the mixture with Klenow Fragment to produce blunt ends,separating the fragments by gel electrophoresis, then purifying the 1240bp kan^(R) fragment from a gel slice. The isolated fragments from pUC19and pUC4K were mixed and ligated with T4 ligase following the protocolof GibcoBRL technical bulletin 15244-2 (Rockville, Md.) to produce“intermediate p1”, an intermediate plasmid carrying pUC19 features and akan^(R) gene. Strain E. coli DH5αMCR was transformed with the ligationmixture following an established protocol (“Fresh Competent E. coliprepared using CaCl₂”, pp. 1.82–1.84, in J. Sambrook, E. F. Fritsch andT. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed. (1989)).Transformants were selected on Luria Broth plates containing 50 μg/ml ofkanamycin.

An oriT site for conjugative transfer was obtained from plasmid pFD288by restricting it with HaeII, converting the single stranded ends toblunt ends by treating the mixture with Klenow Fragment, separating thefragments by agarose gel electrophoresis, then purifying the 778 bp oriTfragment from a gel slice. The intermediate p1 plasmid was opened at asingle site with restriction enzyme SapI, then treated with KlenowFragment to convert the single stranded ends to blunt ends. The SapIdigested, blunt ended p1 intermediate was mixed with the purified oriTfragment, then treated with T4 ligase as above to create “intermediatep2”, a plasmid which carries oriT in addition to kan^(R) and otherpUC19-derived features. Strain E. coli DH5αMCR was transformed with theligation mixture as above. Intermediate p2 was confirmed by restrictiondigestion analysis.

The polylinker in intermediate plasmid p2 was replaced with thepolylinker from pFC5. To do this the two plasmids were separatelyrestricted with PvuII, the fragments from each digestion reaction wereseparated by gel electrophoresis, then the pFC5-derived polylinkerfragment (531 bp), and the larger non-polylinker fragment fromintermediate p2 were purified from gel slices. The recovered p2 fragmentand the pFC5-derived polylinker fragment were mixed and joined using T4ligase as above to make plasmid pJND1000. The structure of pJND1000 wasconfirmed by restriction digestion analysis. PJND1000 replicates in E.coli, has a functioning kanamycin resistance gene, an RK2-derived oriTsite to permit conjugative transfer into Ketogulonigenium and otherhosts, a polylinker for DNA cloning, forward and reverse M13 primers tofacilitate DNA sequencing reactions into the polylinker, and permitsscreening for cloned inserts by inactivation of lacZα using Xgalindicator plates.

Example 5 Isolation of Plasmid pXB1 Containing LinearizedKetogulonigenium Plasmid pADMX6L1

The DNA sequence of plasmid pADMX6L1 (SEQ ID NO:1) is about 7029 bp longand contains a single BamHI restriction site. The BamHI site wasutilized to clone the pADMX6L1 sequence into the E. coli vectorpJND1000. A DNA prep containing a mixture of the endogenous plasmidsfrom Ketogulonigenium robustum ADMX6L, and purified pJND1000 DNA from E.coli, were made as in Example A and separately digested with restrictionenzyme BamHI. A Wizard DNA Clean-Up kit was used to separate thedigested DNA from enzyme and salts in preparation for the next step,with a final DNA suspension volume of 50 μl. DNA from the two digestions(3 μl of pJND1000 DNA and 10 μl of Ketogulonigenium plasmid DNA) wasmixed and ligated overnight at room temperature using T4 ligase with theprotocol of GibcoBRL technical bulletin 15244-2 (Rockville, Md.). StrainE. coli DH5αMCR was transformed with the ligation mixture following anestablished protocol (“Fresh Competent E. coli prepared using CaCl₂”,pp. 1.82–1.84, in J. Sambrook, E. F. Fritsch and T. Maniatis, MolecularCloning: A Laboratory Manual, 2nd Ed. (1989)). Transformants were spreadon Luria Broth agar plates containing 50 μg/ml of kanamycin and 40 μg/mlof Xgal and grown at 37 deg C. Colonies that were white, indicatinginsertion of DNA into the pJND1000 BamHI site, were picked and culturedfor further processing. Digestion of plasmid DNA from transformantsusing BamHI, EcoRV, and XhoI separately, yielded the expected fragmentsizes for sucessful cloining of pADMX6L1 into the pJND1000 E. colivector. The identity of the pADMX6L1 insert was further confirmed bypartial DNA sequencing into the pADMX6L1 DNA region using the M13sequencing primer regions of the pJND1000 vector. The chimeric plasmidcontaining linearized pADMX6L1 cloned into pJND1000 was named pXB1. AnE. coli host transformed with pXB1 was deposited in the patentcollection of the National Regional Research Laboratories in Peoria,Ill., U.S.A. under the terms of the Budapest Treaty as NRRL B-30418.

Example 6 Isolation of Plasmid pXH2 Containing LinearizedKetogulonigenium Plasmid pADMX6L2

The DNA sequence of plasmid pADMX6L2 (SEQ ID NO:2) is about 4005 bp longand contains a single HinDIII restriction site. The HinDIII site wasutilized to clone the pADMX6L2 sequence into the E. coli vector pUC19.The procedure was the same as in Example 5, except that prior to theligation step the plasmid DNAs were digested with HinDIII instead ofwith BamHI, and transformants were plated on LB agar plates containingXgal and 100 μg/ml of ampicillin instead of the kanamycin. Plasmid DNAfrom transformants giving white colonies was isolated and digested withHinDIII and NdeI, giving the expected fragment sizes for correctinsertion of linearized pADMX6L2 DNA into the pUC19 vector. The identityof the pADMX6L2 insert was further confirmed by partial DNA sequencinginto the pADMX6L2 region using the M13 primer regions of pUC19. Thechimeric plasmid containing linearized pADMX6L2 cloned into pUC19 wasnamed pXH2. An E. coli host transformed with pX-H2 was deposited in thepatent collection of the National Regional Research Laboratories inPeoria, Ill., U.S.A. under the terms of the Budapest Treaty as NRRLB-30419.

Example 7 Construction of E. coli/Ketogulonigenium Shuttle PlasmidpXH2/K5

Since kanamycin resistance can be expressed in Ketogulonigenium, it wasdesireable to replace the ampR resistance gene in pXH2 with a kanamycinresistance gene, thereby allowing the selective isolation ofKetogulonigenium strains transformed with a plasmid. In vitrotransposition was used to move a kanamycin resistance gene into pXH2 andsimultaneously inactivate ampicillin resistance. Insertion of akanamycin resistance gene into the ampicillin resistance gene of pXH2was achieved using Epicentre technologies EZ::TN Insertion System(Madison, Wis.). 0.05 pmoles of pXH2 was combined with 0.05 pmoles ofthe <KAN-1> Transposon, 1 μl of EZ::TN 10X Reaction Buffer, 1 μl ofEZ::TN Transposase, and 4 ul of sterile water giving a total volume of10 μl in the transposition reaction. This mixture was incubated at 37°C. for 2 hours, then stopped by adding 1 μl of EZ::TN 10X Stop Solution,mixing, and incubation at 70° C. in a heat block for 10 minutes. E. coliDH5αMCR was transformed with the DNA mixture and transformants wereselected on Luria Broth agar plates containing 50 μg/ml of kanamycin.Colonies recovered from these plates were patched onto two LB agarplates, one with 50 μg/ml of kanamycin and the other with 100 μg/ml ofampicillin. Only those that grew on the kanamycin plates were saved.Plasmid DNA from an ampicillin sensitive, kanamycin resistant colony wasisolated as “pXH2/K5”. The proper insertion of the kanamycin resistancetransposon into the vector ampicillin resistance gene was confirmed byanalyzing pXH2/K2 DNA with various restriction endonucleases. Todemonstrate the viability of pXH2/K5 as an E. coli/Ketogulonigeniumshuttle vector, Ketogulonigenium robustum strain ADMX6L was transfromedwith pXH2/K5 DNA using the electroporation technique of Example 2.Stable, kanamycin resistant transformants were obtained from whichpXH2/K5 DNA can be reisolated.

Example 8 Isolation of Plasmid pXB4 Containing LinearizedKetogulonigenium Plasmid pADMX6L4

The DNA sequence of plasmid pADMX6L4 (SEQ ID NO: 4) is about 4211 bplong and contains a single SspI restriction site. The SspI site wasutilized to clone the pADMX6L4 plasmid into the E. coli vector pUC19.The procedure was the same as in Example 6, except that prior to theligation step the plasmid DNA from strain ADMX6L was digested with SspIand pUC19 was digested with SmaI. Plasmid DNA from transformants givingwhite colonies was isolated and double digested with EcoRI and BamHI,and separately with PstI and SphI, in each case giving the expectedfragment sizes for correct insertion of linearized pADMX6L4 DNA intopUC19. Partial DNA sequencing into the pADMX6L4 region using the M13primers of pUC19 further confirmed the identity of the pADMX6L4 insert.The chimeric plasmid containing linearized pADMX6L4 cloned into pUC19was named pXB4. An E. coli host transformed with pXB4 was deposited inthe patent collection of the National Regional Research Laboratories inPeoria, Ill., U.S.A. under the terms of the Budapest Treaty as NRRLB-30435.

Example 9 Definition of DNA Sequences Comprising Replication Functionsof Ketogulonigenium Plasmids

Various programs of the Wisconsin Package version 10.1 (GeneticsComputer Group, Inc. Madison, Wis.) were used to analyze the DNAsequences of the four endogenous plasmids from Ketogulonigenium strainADMX6L. The analysis revealed multiple open reading frames (ORFs),nucleotide sequences having protein-encoding potential, on each plasmid.The predicted amino acid sequences of these ORFs was obtained, and asimilarity search against PIR and SWISS-PROT protein databases wasconducted using the GCG implementations of the FASTA (Proc. Natl. Acad.Sci. USA 85:2444–2448 (1988)) and BLAST (Altschul et al., Nucleic AcidsResearch 25:3389–3402 (1997)) methods. Based on sequence similarity toknown plasmid-encoded replication proteins, plasmids pADMX6L1, pADMX6L2,and pADMX6L3 were found to encode potential plasmid replicationproteins. The genes (ORFs) for these functions have the followingendpoints:

pADMX6L1 rep ORF: bases 2255–2710 of FIG. 1 (SEQ ID NO:1) pADMX6L2 repORF: reverse compliment of bases 3960–2562 of FIG. 2 (SEQ ID NO:2)pADMX6L3 rep ORF: reverse compliment of bases 5031–4003 of FIG. 3 (SEQID NO:3).

A region of DNA sequence upstream and downstream of the regions definedby these ORFs, perhaps 500 bp in each direction, is likely to containtranscriptional promoters, terminators, and other sequences required forproper expression of the replication proteins and control of plasmidreplication. Therefore a region comprising part or all of the plasmidreplicon for these three plasmids could be defined by the following DNAsequences:

pADMX6L1 replicon: bases 1755–3210 of the FIG. 1 sequence, alsorepresented by SEQ ID NO:5 pADMX6L2 replicon: bases 455–2060 of the FIG.2 sequence, also represented by SEQ ID NO:6 pADMX6L3 replicon: bases3503–5531 of the FIG. 3 sequence, also represented by SEQ ID NO:7

All publications mentioned herein above are hereby incorporated in theirentirety by reference. All publications cited in the annotations to theGenbank accession numbers or in ATCC strain descriptions cited hereinare hereby incorporated in their entirety by reference.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

1. An isolated or purified nucleic acid molecule comprising a polynucleotide comprising a replicon functional in Ketogulonigenium, the polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 4; (b) the nucleotide sequence of the Ketogulonigenium portion of the plasmid contained in NRRL Deposit No. B-30435; (c) the nucleotide sequence that is the complement to SEQ ID NO: 4; and (d) the nucleotide sequence that is the complement to the Ketogulonigenium portion of the plasmid contained in NRRL Deposit No. B-30435.
 2. The isolated or purified nucleic acid molecule of claim 1, wherein said isolated or purified nucleic acid molecule has a nucleotide sequence that is at least 95% identical to that of part (a).
 3. The isolated or purified nucleic acid molecule of claim 1, wherein said isolated or purified nucleic acid molecule has a nucleotide sequence that is that of part (a).
 4. The isolated or purified nucleic acid molecule of claim 1, wherein said isolated or purified nucleic acid molecule has a nucleotide sequence that is at least 95% identical to that of part (b).
 5. The isolated or purified nucleic acid molecule of claim 1, wherein said isolated or purified nucleic acid molecule has a nucleotide sequence that is that of part (b).
 6. The isolated or purified nucleic acid molecule of claim 1, wherein said isolated or purified nucleic acid molecule has a nucleotide sequence that is at least 95% identical to that of part (c).
 7. The isolated or purified nucleic acid molecule of claim 1, wherein said isolated or purified nucleic acid molecule has a nucleotide sequence that is that of part (c).
 8. The isolated or purified nucleic acid molecule of claim 1, wherein said isolated or purified nucleic acid molecule has a nucleotide sequence that is at least 95% identical to that of part (d).
 9. The isolated or purified nucleic acid molecule of claim 1, wherein said isolated or purified nucleic acid molecule has a nucleotide sequence that is that of part (d).
 10. A vector comprising the nucleic acid molecule of claim 1 and at least one marker gene.
 11. The vector of claim 10, wherein said marker gene confers resistance to an antibiotic selected from the group consisting of amikacin, ampicillin, chloramphenicol, erythromycin, gentamicin, kanamycin, penicillin, spectinomycin, streptomycin, and tetracycline.
 12. A vector comprising: (a) the nucleic acid molecule of claim 1; (b) a terminator of transcription; (c) a promoter; and (d) a discrete series of restriction endonuclease recognition sites, said series being between said promoter and said terminator.
 13. The vector of claim 12, comprising a replicon functional in an organism of the genus selected from the group consisting of Acetobacter, Corynebacterium, Rhodobacter, Paracoccus, Roseobacter, Pseudomonas, Pseudogluconobacter, Gluconobacter, Serratia, Mycobacterium, Streptomyces and Bacillus.
 14. The vector of claim 12, comprising a replicon functional in Escherichia coil.
 15. A transformed cell of Escherichia coil comprising the vector of claim
 14. 16. A transformed cell of the genus Ketogulonigenium comprising the vector of claim
 14. 17. An isolated nucleic acid molecule comprising a polynucleotide comprising a replicon functional in Ketogulonigenium, wherein the polynucleotide which hybridizes under stringent hybridization conditions to a sequence selected form the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 4; (b) the nucleotide sequence of the Ketogulonigenium portion of the plasmid contained in NRRL Deposit No. B-30435; (c) the nucleotide sequence that is the complement to SEQ ID NO: 4; and (d) the nucleotide sequence that is the complement to the Ketogulonigenium portion of the plasmid contained in NRRL Deposit No. B-30435; wherein said polynucleotide which hybridizes, does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. 